1. Field of the Invention
This invention relates to improvements in obtaining medical diagnoses and in particular to a novel one-stage prothrombin assay.
2. Description of the Prior Art
The clotting of blood requires the participation of approximately thirteen clotting factors in a sequence of reactions whereby certain factors are in turn activated by preceding factors or a combination of preceding factors, often in conjunction with various accessory factors such as calcium ion and negatively charged phospholipid bilayer surface. It is known that clotting proceeds by at least two pathways: the so-called extrinsic and intrinsic pathways. The former is initiated by the release of thromboplastin from damaged tissue, while the latter is initiated by contact activation of Factor XII. Both pathways can be accelerated by a feedback loop via thrombin activation on at least Factors VIII and V. The clotting mechanism is represented in simplified form by the following diagram A, in which Roman numerals identify the various clotting factors. ##STR1##
In order to screen for deficiencies in blood factors of either genetic or disease origin and to monitor anticoagulant therapy, three common tests are employed. All three tests measure the time required for the clotting of a patient's plasma upon addition of certain reagents. Each test determines the effectiveness of a portion of the clotting mechanism and thus allows judgements with respect to the effectiveness of the blood factors involved in the studied portion or with respect to the result of anticoagulant therapy.
The first test is the partial thromboplastin time test (PTT), which evaluates the intrinsic pathway. The second test is the one-stage prothombin time test (herein "prothrombin time" or PT), which evaluates the extrinsic pathway. The third test is the thrombin time test (TT), which evaluates that portion of the clotting mechanism subsequent to thrombin production, which is generally dependent upon both fibrinogen concentration and fibrin polymerization. The collective results of these three tests, together with repetitions of the tests in substitution studies (if necessary) allow determination of precise factor deficiencies. It should be noted that a prolongation of the thrombin time invariably also leads to a prolongation of both the partial thromboplastin time and the prothrombin time as currently measured, since a defect subsequent to thrombin production prolongs clotting time regardless of how the clotting mechanism is initiated. A significant deficiency in this portion of the clotting mechanism is, however, quite rare compared to deficiencies in other clotting factors.
The above discussion having been presented as a general background in the mechanism of coagulation, attention will now be focused on the prothrombin time test.
The prothrombin time test is a valuable and widely used diagnostic tool employed to monitor oral anticoagulant (e.g., dicoumarol) therapy and to screen for genetic or disease-caused deficiencies in blood factors I, II, V, VII, and X. This test, as with the other two clotting time tests, is based upon the length of time required for a patient's plasma to clot under the influence of certain reagents--for the PT test these reagents are calcium ion and thromboplastin. The process is illustrated schematically as follows (Diagram B): ##STR2##
It can thus be seen that the prothrombin time is a function of the times required for the two processes depicted above. The first of these times is the thrombin generation time, that is, the length of time required for the formation of effective amounts of thrombin from prothrombin. The second time is the gelation time, that is, the time required for the actual clot to form once sufficient thrombin is present to promote the reaction of fibrinogen to fibrin. The gelation time is negligible in the presence of excess thrombin concentration compared to fibrinogen concentration for a fibrinogen-normal individual (as defined below). The prothrombin time test is sensitive to only major defects in the second step in Diagram B, due to the large excess of thrombin produced.
The prothrombin time test as presently performed measures the time required for the occurrence of a physical event--clotting, which is to be distinguished from a measurement of chemical concentration or rate. A variety of physical phenomena have been recognized in the art as endpoints for clotting time tests, such as turbidity (of various degrees), precipitates, appearance of strands, change in surface tension, and the like. A variety of instruments have been developed, using different endpoint recognition criteria, so that measurements among instruments are not readily comparable. Moreover, the prothrombin time measurement of the prior art does not lend itself very well to automation, which is a highly desirable objective in modern day diagnostic medicine. While there are instruments for determining prothrombin time which are fully automated, these are generally based on a turbidity measurement to evaluate the formation of a clot and provide a measurement expressed in terms of time.
A further practical difficulty with the current prothrombin time test is that thromboplastin form various commercial sources does not always yield comparable results with the same plasma samples. That is, one cannot quantitatively predict the PT times for a group of plasma samples using thromboplastin from one source based on PT times obtained for the same group of plasma samples using thromboplastin from another source. In other words, it is not possible (for a group of plasma samples) to correlate quantitative PT test results using one source of thromboplastin with those using another source of thromboplastin. This lack of correlatability is recognized in the art as a serious deficiency with the conventional prothrombin time test.
In a typical prothrombin time test as it is currently performed, 0.2 ml of thromboplastin containing calcium ions is added to 0.1 ml of citrated plasma, and the time interval from the moment of addition to the first indication of a clot is measured. Prothrombin times for normal plasmas range from 10 to 14 seconds depending on the laboratory and the particular method employed. For example, optical density instruments tend to give times of 10-11 seconds while manual methods and measurements on a Fibrometer brand clotting instrument tend to give 12-14 seconds for normal plasmas. Clotting times higher than these indicate that the plasma has a deficiency in concentrations of fibrinogen (Factor I), prothrombin (Factor II), Factor V, Factor VII, or Factor X, or a combination of these. This abnormal condition may be caused either by genetic deficiency, by disease, or by anticoagulant therapy. The term "abnormal plasma" as used herein specifically includes plasma of individuals undergoing anticoagulant therapy, as well as those having deficiencies of pathological or genetic origin.
For example, patients to whom heparin (a direct anticoagulant) is being administered may have lengthened clotting times because heparin accelerates the inhibition of thrombin formation and therefore slows down the fibrinogen-to-fibrin reaction catalyzed by thrombin. On the other hand, indirect anticoagulants such as coumarin and dicoumarol inhibit the synthesis of Factors II, VII, and X. (Factor IX is also inhibited by these indirect anticoagulants, but this factor is not part of the extrinsic pathway initiated by thromboplastin.) The subnormal quantities of any of these non-thrombin factors in turn inhibit thrombin formation with consequent reduction in the fibrin clotting reaction and prolonged times in the prothrombin time test. Prothrombin times for abnormal plasma can be as long as about 60 seconds as the test is currently performed.
It is now known that at least the portion of the coagulation mechanism which is common to both the intrinsic and extrinsic pathway proceeds by proteolytic action in which prothrombin is cleaved by a combination of activated Factors V and X in combination with calcium ion and a negatively charged phospholipid bilayer surface to produce thrombin and other fragments. Thrombin in turn acts as a proteolytic enzyme to cleave fibrinogen into fibrin, and a clot is formed.
Knowledge of the mode of action of these clotting factors has led to the development of chromogenic substrates for their determination. These chromogenic substrates are materials which are acted upon and cleaved by certain clotting factors with the resultant production of a colored material; hence the name "chromogenic" (color producing) "substrate" (material acted upon by an enzyme). Since the substrate is designed so that the absorbance maximum for the uncleaved substrate is distinctly different from that of the chromophore released by cleaving process, the determination of the concentration of the cleaving clotting factor reduces to a simple absorbance measurement either at the maximum absorption point of the cleaved chromophore or some other convenient distinguishing wavelength.
These chromogenic substrates generally comprise a polypeptide portion and a chromophore portion; the polypeptide portion can be tailored to be specific to a desired clotting factor, which will then cleave the substrate between the chromophore and the peptide, thus releasing the chromophore. The chromophore is typically p-nitroaniline (pNA) but may also be any of a variety of other materials such as 4-methoxy-.beta.-naphthalene, 4-methoxy coumarin, 4-trifluoromethyl coumarin, and the like. Polypeptide portions which may be used for the detection and thrombin include L-phenyl-alaninyl-L-pipecolyl-L-arginyl, L-phenylalaninyl-L-valyl-L-arginyl, glycyl-L-prolyl-L-arginyl, and the like. The amino terminus of the polypeptide portion is usually blocked by a blocking group, such as benzoyl, carboxyl, tosyl, a D-amino acid residue, or the like, while the chromophore is attached to the carboxyl terminus. Included within the term "chromogenic substrates" are those materials wherein the cleaved chromophore is detected by its emission spectrum (e.g., fluorescence) such as 4-methoxy-.beta.-naphthalene, as well as those wherein the cleaved chromophore is detected by its absorbance spectrum such as p-nitroaniline. Chromogenic substrates are known which are specific for thrombin.
Despite the existence of chromogenic substrates for the detection of thrombin, no practical method using these substrates for the evaluation of the single-stage prothrombin time is known. It is believed that this lack is occasioned by following heretofore unsolved problems.
In order to employ the chromogenic substrate in the evaluation of (e.g.) prothrombin time, it is necessary to use a highly diluted test plasma. Otherwise, the formation of the clot totally obscures the color production and renders impossible the measurement of any color change. Moreover, the color produced in undiluted plasma would be too intense for meaningful measurement of color change to be made. If dilute plasma is used, an abnormal prothrombin time can extend to as long as ten minutes or longer, while a normal prothrombin time is extended to about 1-2 minutes. These thrombin generation times are too long to be of practical use, especially in an automated determination method. They are also too disparate in length to both be conveniently measured on the same commercially-available spectrophotometer because of the built-in limitations of such instruments. While it is recognized that some improvements have been made in this area, nevertheless, the determination of prothrombin times using chromogenic substrates has not heretofore been practically possible.